PROJECT SUMMARY/ABSTRACT Several forms of neurodegeneration have been linked to perturbations in RNA homeostasis. In the case of pontocerebellar hypoplasia type 10 (PCH10), neurodegeneration is due to homozygous mutation of the RNA kinase CLP1 (cleavage and polyadenylation factor I subunit 1), which phosphorylates the 5?OH of RNA. PCH10 is a rare pediatric disorder resulting in profound cognitive impairment, motor dysfunction, and epilepsy as a result of hypoplasia and/or atrophy of the brain and spinal cord. The causative mutation in CLP1 (R140H) leads to partial loss of function; however, why this results in neurodegeneration is unclear. In vitro experiments suggest that CLP1 may play roles in mRNA cleavage, tRNA splicing, and miRNA activation; however, at present, there is not enough evidence to conclude that it performs any of these functions in vivo. In order to discover the functions of CLP1 implicated in PCH10, we have generated a knock-in mouse line with the patient mutation that displays mild motor defects. We have also generated a Clp1 null allele which, in combination with the patient mutation allele, produces a severe motor phenotype in Clp1R140H/- mice. Our preliminary data demonstrates that Clp1R140H/- mice have degeneration of lower motor neurons. This proposal expands upon this finding in two ways. In Aim 1, we propose to extend our analysis of the Clp1R140H/- phenotype to the brain and sensory neurons. This will clarify which subtypes of neurons are sensitive to Clp1 mutation. We will first perform histological analyses to identify any gross abnormalities, and then focus specifically on cells and brain regions affected in patients. In Aim 2, we will examine the molecular phenotype associated with Clp1 mutation using FACS-sorted motor neurons from mutant and wild-type mice to make libraries for RNA profiling. CLP1 is a member of the mRNA polyadenylation and cleavage complex. Thus we will investigate the role of CLP1 in mRNA cleavage by generating 3? end mRNA libraries. This will indicate whether loss of CLP1 alters mRNA cleavage and whether this occurs in a sequence-specific or -independent manner. Second, we will use a set of miRNA libraries to determine whether CLP1 phosphorylates miRNAs in motor neurons. While most miRNAs are thought to be constitutively active, it has been demonstrated that one miRNA is maintained in a dephosphorylated state until application of a stimulus prompts 5? phosphorylation by CLP1. In conclusion, by exploring the functions of CLP1 in mRNA and miRNA processing in motor neurons in a mouse model of PCH10, we will elucidate the mechanism(s) by which Clp1 mutations lead to neurodegeneration.